Dextran sieving

Carbon membranes were fabricated by pyrolysis of a mixture of polyffurfuiyl alcohol) (PFA) and poly(ethylene glycol) (PEG) that was spin-coated on a mac-roporous stainless steel support. The stainless steel support was first modified by physical deposition of sub-micron-sized silica particles within the micropoies and then PFA/PEG/acetone solution was spin-coated onto the silica-modified support. Coated support was heated in a stream of Ar to 600°C for 4 h for pyrolysis. The process was repeated. Hydrauhc permeability was obtained under 40-50 psig. UF experiments with blue dextran as the solute was also condueted. Table 6.1 shows the hydraulic permeability and blue dextran sieving data. 

Dextran sieving curves were also evaluated by filtration of dextrans of different average molecular weights in kDa, i.e. 2000,167,75,40, and 10 kDa. Sieving coefficient versus dextran average molecular weight is given in Fig. 6.1. 

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

If the soluble protein that specifically adsorbs to the fiber can be extrinsi-cally labeled, the background problem can be avoided. Of course, in vivo proteins cannot be labeled. However, it is conceivable that a protein labeled with a bulky extrinsic group (e.g., fluorescent dextrans) could be confined by a molecular sieve membrane (e.g., a dialysis membrane) within a closed volume surrounding the specifically derivatized optical fiber. When exposed to the (unlabeled) protein in the biological fluid under investigation, the membrane-clad fiber would allow some unlabeled protein to permeate in and... 

Porath, J. Cross-linked dextrans as molecular sieves. Advances in Protein Chcmistiy 17, 209—226 (1962). 

A good separation of L-rhamnose, 2-acetamido-2-deoxy-D-glucose, D-glucose, and 2-amino-2-deoxy-D-glucose, eluted in that order, was obtained by Zeleznick80 on a small column packed with the highly cross-linked, dextran gel Sephadex G-25, with 62 15 25 (v/v) butyl alcohol-M acetic acid-water as the eluant however, the gel functioned more as a support for partition chromatography than as a molecular sieve. 

Dextran gels used as molecular sieves are formed by crosslinking dextrans with epichlorhydrin to give a semisynthetic polysaccharide with a well-defined pore size (Sephadex ). 

The molecular-sieve dextran gels are widely used in chemistry, biochemistry, and pharmacy for the analytical and preparative separation of metabolites and other biological products. 

Extrapolated from data on lower MW dextrans, or inferred from results with larger particles, e.g, viruses. h Column packed with sieved glass. 

When plotting the log of the reduced mobility vs. the log of the molecular mass of the analyte the typical sigmoidal curves known from DNA analysis are also obtained. The concentration of the sieving polymer has to be beyond its entanglement threshold concentration c. A typical plot is shown in Fig. 10, where different concentrations of dextran T 2000 have been added to the running buffer. Also included are the reduced mobilities of the PSS in the plain buffer. As can be seen, the sieving properties are improved with increasing dextran concentra-... 

It should always be kept in mind that the analysis time increases with increasing concentration of the sieving media. The regime where a separation is no longer possible seems to drift to lower molecular masses. This has been observed with PEG and dextrans (cf. Fig. 10). 

In Fig. 11 the influence of the molecular mass of the sieving medium is demonstrated. There is no difference between dextran T 500 and T 2000 at identical concentration. Less efficient separations are only observed with dextran T 70. The mass concentration here is just around the overlap threshold. For practical reasons it is advisable to use the sieving polymer which gives the lowest viscosity in the buffer solution at the required concentration. 

With PVA-coated capillaries [6], it was possible to separate at pH 2.5 the polyelectrolyte 2-polyvinylpyridinium hydrochloride (2-PVP) in the molecular mass range between 1.5 and 1730 kDa with dextran as sieving matrix [26]. A separation of standards is depicted in Fig. 13. Comparing the efficiencies of the... 

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